1. Field of the Invention
This invention relates to a thyroid immunoassay and, in particular, to a thyroxine (T.sub.4) and triiodothyronine (T.sub.3) polarization fluoroimmunoassay.
2. Description of the Prior Art
The assessment of thyroid status in vitro requires the measurement of circulating thyroid hormone levels, notably, thyroxine (T.sub.4) and triiodothyronine (T.sub.3), which previously was accomplished by methodologies such as protein bound iodine. With the advent of competitive protein binding (CPB) assays, the method of Murphy et al. (1) became in vogue for the estimation of T.sub.4 in serum. In the Murphy et al. (1) methodology, T.sub.4 is extracted from serum with ethanol or a mixture of ethanol and butanol before analysis via a CPB assay employing thyroxine binding globulin (TBG) as a binder. In order to alleviate the extraction and evaporation steps, Braverman et al. (2) absorbed serum T.sub.4 supplemented with radiolabeled hormone onto a small bead-formed dextran gel (sold under the trademark Sephadex) column at pH 12-13. In the alkaline media T.sub.4 is released from serum proteins and adsorbed onto the gel. Because the serum components are not retained by the column, a CPB analysis can be performed on the bead-formed dextran gel column using a fixed amount of TBG. The column also serves to separate free and TBG bound T.sub.4. Although the column-CPB procedure eliminated the cumbersome extraction and separation steps, a major disadvantage remained in that new columns had to be prepared or purchased for each new test. Finally, Alexendar et al. (3) eliminated this problem by regenerating the columns after each cycle with excess TBG.
Presently, radioimmunoassay (RIA) is the method of choice for the determination of serum T.sub.4. The reason for this is that RIA is simpler, quicker, and more specific than the corresponding CPB assays (4, 5). However, a RIA determination in unextracted serum requires the addition of agents to the reaction mixture to prevent the binding of T.sub.4 by endogenous serum proteins. Although salicylates (6) and thiomersal (7) have been employed, the most effective reagent has been 8-anilino-1-naphthalene sulfonic acid (ANS) (4). In addition to the direct RIA approach, the beforementioned column methodology has also been applied to give a RIA for serum T.sub.4 (8).
Sterling et al. made the interesting observation that thyrotoxicosis may be due to an elevated serum T.sub.3 concentration associated with a normal serum T.sub.4 level and that a eumetabolic state could be maintained with a low T.sub.4 if the serum T.sub.3 concentration was within normal limits (9). Although the serum concentration of T.sub.3 is only 1-2% of T.sub.4, its vital role in thyroid physiology is now generally accepted. Serum T.sub.3 concentrations either by saturation analysis after paper chromatography (10) or by gas chromatography (11) are inaccurate, primarily because T.sub.4 is deionated to T.sub.3 during these procedures. T.sub.3 analysis by direct RIA of serum requires (as in the case of direct RIA for T.sub.4) the addition of a blocking agent, usually ANS, that prevents binding by endogenous serum proteins (12, 13). Finally, in a similar fashion to the column approach to T.sub.4, an RIA methodology has also been described for serum T.sub.3 which employs small, reusable bead-formed dextran gel columns (14).
Both an homogenous enzyme immunoassay (EMIT) as well as a more conventional enzyme linked immunoabsorbent assay (ELISA) have been employed for the measurement of serum T.sub.4 (15, 16). Although Smith (17) described the preparation of a fluorescent derivative of T.sub.4 whose fluorescence is enhanced when bound by anti-T.sub.4 serum, this principle cannot be directly applied for the measurement of serum T.sub.4 concentration due to the appreciable and variable levels of intrinsic serum fluorescence. A more conventional approach to a fluorometric immunoassay (FIA) for both T.sub.4 and T.sub.3 based on the separation of free and antibody-bound fractions of labeled ligand, in direct analogy to RIA has been reported by Curry et al. (18). The use of a heterogeneous assay removed most of the serum sample components that might interfer with the measurement of fluorescence from the antibody-bound tracer.
Recently, Schroeder et al. (19) developed an immunoassay for serum T.sub.4 that was monitored by chemiluminescence. In this heterogeneous immunoassay, columns absorbed both sample T.sub.4 and the T.sub.4 -chemiluminescent labeled conjugate, while serum components and potential interferents were washed through the column with buffer. After the addition of antibody and an incubation period, the antibody bound T.sub.4 was eluted and the labeled hormone detected by a H.sub.2 O.sub.2 -microperoxidase chemiluminescence assay.
Although the basis for fluorescence polarization immunoassay has been described and demonstrated (20), to date other than for the fluorescence polarization assays of gentamicin (21) and dilantin (22), this methodology has not been applied in practical assays of proven clinical utility. For example, in the case of enhancement fluoroimmunoassays (17), it is reported that the variable levels of intrinsic serum fluorescence present an obstacle to the assay of patient sera samples.